Development of sheep embryos in vitro in a medium supplemented with different batches of serum albumin

Variability in different lots of commercial serum albumin affects mammalian embryo development in culture. The composition of commercial preparations of ovine, bovine and defatted bovine serum albumin and a fraction of ovine serum containing proteins with a mean molecular weight of 65 kDa (fraction 3) was examined by polyacrylamide gel electrophoresis. All preparations were heavily contaminated with serum proteins other than albumin. Day-6 sheep morulae were cultured for 48 h in a basal bicarbonate-buffered salt solution supplemented with the commercial preparations of ovine, bovine or defatted bovine serum albumin. These three albumin preparations differed in their abilities to support the development of morulae into expanded blastocysts, but these differences disappeared when the basal medium was also supplemented with a component of ovine serum containing substances with molecular weights of less than 10 kDa. In the latter case, the three commercial albumin preparations and fraction 3 of ovine serum all supported full development in about 40-60% of morulae.     

The time-course development of the effects of ethanol ingestion on choline oxidase

Male rats were fed a low-fat diet containing 36% of calories as ethanol, and the time-course development of the effects of ethanol on liver mitochondrial oxidation of choline was determined. Ethanol induced an increase in choline oxidase at days 2, 5 and 7 after being introduced into the diet. Due to an observed 32% increase in total fatty acids in the whole liver, defatted bovine serum albumin was added to the buffer used to homogenize the liver. The presence of bovine serum albumim resulted in a significant decrease in choline oxidase activity at days 2 and 5; however, ethanol still induced an increase in choline oxidase activity in these mitochondria. The total fatty acid concentration of mitochondria prepared in the absence of bovine serum albumin increased steadily until day 5; however, by day 7 the fatty acid concentration had returned to control levels. The addition of bovine serum albumin to the homogenization medium prevented the increase in the total amount of fatty acids. The fatty acid composition of the bovine serum albumin-treated mitochondria, however, was not different from the mitochondria is isolated in the absence of bovine serum albumin. Further, the addition of a free fatty acid to isolated mitochondrial preparations caused about a 100% increase in choline oxidase. These data are consistent with the idea that choline oxidase may be regulated to some extent by an influx or an increase in free fatty acids in the liver as a result of ethanol ingestion. Thus, a second mechanism has been described which contributes to the increase in choline oxidase after ethanol ingestion.     

Amylose-like polysaccharide accumulation and hyphal cell-surface structure in relation to citric acid production by Aspergillus niger in shake culture

When 120 mg glucose/ml was used as a carbon source, in shake culture Aspergillus niger Yang no. 2 maximally produced only 15.4 mg citric acid/ml but accumulated 3.0 mg extracellular polysaccharide/ml. The polysaccharide secreted by mycelia of Yang no. 2 in shake culture was confirmed to be an amylose-like alpha-1,4-glucan by hydrolysis analysis with acid, amylase and glucoamylase. However, in static cultures, such as semisolid and surface cultures free from physical stresses caused by shaking damage, Yang no. 2 produced more citric acid but did not accumulate the polysaccharide. With cultivation time in shake culture, the amount of extracellular polysaccharide and the viscosity of the culture broth increased. The increase of shaking speed caused a remarkable increase in the accumulation of extracellular polysaccharide, e.g. 11.2 mg extracellular polysaccharide/ml was accumulated in the medium at a shaking speed of 200 rpm. The addition of 2.0 mg carboxymethylcellulose (CMC)/ml as a viscous additive to the medium reduced drastically the amount of extracellular polysaccharide accumulated to 1.5 mg/ml, but increased the citric acid produced to 52.0 mg/ml. However, intracellular polysaccharide accumulation kept up a steady rate of 0.26 microgram/mg dried mycelium through the entire period of cultivation. The addition of 3.0 mg polysaccharide/ml purified from the culture broth to the medium at the start of a culture resulted in a decrease of extracellular polysaccharide accumulation but an increase of citric acid accumulation. From electronmicroscopic observation, cell surfaces of hyphae cultivated with CMC were smooth, while hyphae cultivated without CMC had fibrous and granular polysaccharide on the cell surface. These results suggested that Yang no. 2 secreted the polysaccharide on the cell surface as a viscous substance and/or a shock absorber to protect itself from physical stresses caused by shaking damage in shake culture.     

Hyaluronate binding proteins also bind to fibronectin, laminin and collagen

Small molecular weight proteins, isolated from the culture medium of embryonic chick heart fibroblasts and 3T3 cell lines by hyaluronate affinity chromatography, bind in order of apparent affinity, to hyaluronate, fibronectin, collagen and laminin. Such proteins isolated from the MSV-transformed 3T3 cell line bind in greater amounts to the nectins and hyaluronate than do similar proteins isolated from heart fibroblasts or 3T3 cells. These small hyaluronate binding proteins are immunologically distinct from other well characterized proteins such as laminin, fibronectin, bovine serum albumin and actin. Their relationship to other small, extracellular proteins and their possible role in structuring of extracellular matrix are discussed.     

Extraction of an erythrotropin-like factor from bovine serum albumin (cohn fraction V)

Summary Different batches of commercially available bovine serum albumin (Cohn fraction V) were tested in a serum-free medium for their ability to stimulate thymidine incorporation in erythroid cells of fetal bovine liver. All preparations stimulated thymidine incorporation. Crystallized, charcoal-treated, or fatty acid-free albumin had substantially lower thymidine incorporation-stimulating activities than the crude preparations. The albumin preparations also had a synergistic effect with respect to erythropoietin on erythroid cells from rat liver, a typical property of erythrotropins. One gram of one of the batches of Cohn fraction V was fractionated by reversed-phase high performance liquid chromatography (HPLC). The fraction with thymidine incorporation-stimulating activity had a similar elution position as erythrotropin isolated from fetal bovine serum. Further purification using reversed-phase HPLC in the presence of trifluoroacetic acid and heptafluorobutyric acid and gel permeation HPLC resulted, in the isolation of a factor that is very similar to fetal bovine serum erythrotropin. It has practically the same specific activity as the purified fetal peptide in the rat liver bioassay. These results suggest that many of the beneficial effects of the albumin preparations added as supplement of serum-free tissue culture media may be due to the presence of erythrotropin-like factors. The work was supported by grants MT-6072 and ME-9031 from the Medical Research Council of Canada. The author is a Chercheur-Boursier of the Fonds de la Recherche en Santé du Quebec.     

Effects of rat serum on gonadotrophin release in vitro

Gonadotrophin release from a monolayer of cultured pituitary cells from the rat was increased by the addition of fresh rat serum or rat serum albumin to the culture medium. Similar effects were obtained when horse, chicken, lamb or porcine serum was used. Fetal bovine serum or bovine serum albumin showed a stimulatory effect on FSH and LH release but, unlike the case with rat serum, a dose-response release could not be demonstrated. In addition, fresh rat serum caused the accumulation of secretory granules in the gonadotrophs. The addition of LHRH to the culture medium also stimulated gonadotrophin release in a dose-response dependent manner. The dependence of the gonadotrophs on LHRH was markedly increased by the addition of 50% fresh rat serum to the culture medium. These results indicate that fresh rat serum contains some unknown factor(s) which 1) stimulates gonadotrophin release, 2) causes an accumulation of secretory granules, and 3) is important for maintaining secretory capacity of gonadotrophs.     

Effect of protein additives on acetylene reduction (nitrogen fixation) by Rhizobium in the presence and absence of soybean cells

The effect of protein additives on acetylene reduction (N(2) fixation) by Rhizobium associated with soybean cells (Glycine max [L.] Merr.) in vitro was studied. Acetylene reduction was promoted on the basal medium supplemented with 1.4 mg of N/ml supplied as aqueous extracts of hexane-extracted soybean, red kidney beans (Phaseolus vulgaris L.), or peas (Pisum sativum L.). Commercial samples of alpha-casein, or bovine serum albumin also promoted acetylene reduction at a concentration of 1.4 mg of N/ml of basal medium, but egg albumin supplying an equal amount of nitrogen to the basal medium completely suppressed acetylene reduction. Autoclaving the aqueous extract of hexane-extracted soybean meal had no effect on its ability to promote acetylene reduction. The presence of 40 mm succinate decreased acetylene reduction with leguminous proteins supplying 1.4 mg of N/ml but promoted acetylene reduction by Rhizobium 32H1-soybean cell associations on media containing alpha-casein, bovine serum albumin, or egg albumin suppling 1.4 mg of N/ml. Similar results were obtained with both cowpea Rhizobium 32H1 and Rhizobium japonicum 61A96. Pure cultures of Rhizobium 32H1 developed acetylene-reducing activity in the presence of soybean extract on basal agar medium and in vermiculite supplied with N-free mineral salts plus crude soybean meal. The results suggest that in certain situations, free living Rhizobium may reduce N(2) under field conditions.     

Cryo-survival and development of bovine blastocysts are enhanced by culture with recombinant albumin and hyaluronan

Recombinant albumin can be used to supplement culture medium for the maturation and fertilization of bovine oocytes and subsequent embryo development to the blastocyst stage. Recombinant albumin was able to support blastocyst development at rates equivalent to that of bovine serum albumin (BSA) supplemented media. Supplementation of media containing recombinant albumin and citrate stimulated blastocyst expansion. Culture with recombinant albumin and citrate significantly increased the ability of the resultant blastocysts to re-expand and hatch following cryopreservation. The further addition of the glycosaminoglycan hyaluronan to the culture medium containing either BSA or recombinant albumin also increased the ability of blastocysts to survive cryopreservation. Inclusion of recombinant albumin and hyaluronan in culture media facilitates the development of physiological defined culture conditions. For bovine embryos this has implications for both research and commercial applications where defined reproducible conditions are desirable.     

Metabolic interactions between animal cells through permeable intercellular junctions.

The isolated perfused rat liver was used to study the degradation of ¹²⁵I-labelled protein supplied in the perfusion medium. Formaldehyde-denatured proteins (human serum albumin, bovine serum albumin and especially rat liver phosphoenolpyruvate carboxykinase (GTP)) were taken up by the liver and degraded at high rates. Native human serum albumin was not degraded at significant rates by the perfused liver, while native phosphoenolpyruvate carboxykinase (GTP) was catabolised at about one-fourth the rate of the denatured enzyme. The degradation rate of denatured human serum albumin increased markedly as protein was added up to 0.7 mg, and more gradually with further increases in added protein. The biphasic nature of concentration dependence probably reflects the contribution of different cell types in the liver. Autoradiographic examination of serial biopsies taken during perfusion of the liver with formaldehyde-denatured, ¹²⁵I-labelled bovine serum albumin showed that at the cellular level the radioactivity was located predominantly in Kupffer and other non-parenchymal cells; and at the subcellular level the radioactivity was largely in endocytic vesicles, lysosomes and occasionally in the sinusoidal spaces. No significant radioactivity was found associated with other cytoplasmic organelles or the nucleus. It is concluded that lysosomes of the non-parenchymal cells are primarily responsible for the degradation of denatured extracellular protein that enters the liver.     

Effect of the absence or presence of various protein supplements on further development of bovine oocytes during in vitro maturation

The evaluation of culture medium for bovine oocytes has progressed toward more defined conditions during the last few years. The main objective of this study was to evaluate different sources of albumin as a protein supplement during in vitro maturation (IVM) of bovine oocytes in synthetic oviduct fluid medium (SOF). The replacement of protein with polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA) was also evaluated. The effect of recombinant human FSH on cumulus expansion and nuclear maturation in SOF containing BSA (BSA-V) or PVP-40 was also studied. Addition of BSA-V during IVM retarded nuclear maturation when compared with addition of PVP-40 or use of SOF alone. The inclusion of different concentrations of BSA-V, fetal calf serum (FCS), or PVA during IVM had no positive effect on the developmental capacity of the oocytes compared with the use of SOF alone with no supplement but significantly decreased the percentage of embryos reaching the morula and blastocyst stages. However, when BSA-V was replaced with purified BSA, BSA that was essentially free of fatty acids, or chicken egg albumin, embryonic development rates were restored. The presence of PVP-40 but not PVP-360 during IVM significantly increased morula and blastocyst production. These results indicate that although SOF alone can support bovine oocyte maturation, a high proportion of morulae and blastocysts can be produced from IVM oocytes cultured in medium containing PVP-40. These studies are the first to show that the effect of FSH on nuclear maturation and cumulus expansion is dependent on substrates present in IVM medium.     

Activation of apoalkaline phosphatase by serum albumin with Zn2+ in rat hepatoma cells.

Reuber rat hepatoma cells (R-Y121B) cultured at 0.5% serum accumulated apoalkaline phosphatase in intact cells. When R-Y121B cells were cultured in the presence of bovine serum albumin, alkaline phosphatase activity increased in the cells, and the associated increase in enzyme activity differed amongst bovine serum albumin preparations. The treatment of bovine serum albumin with activated charcoal not only enhanced the effect of serum albumin on alkaline phosphatase activity, but also cancelled the differences due to different preparations of serum albumin. In contrast, no effect from serum albumin was observed in the increase of alkaline phosphatase activity in R-Y121B cell homogenates incubated at 37 degrees C. The activated-charcoal treatment of bovine serum albumin increased the amount of Zn2+ bound to the protein. When R-Y121B cells were cultured with bovine serum albumin, the concentration of Zn2+ in the cytosol fraction slightly increased. However, the effect of serum albumin on Zn2+ concentration in the cytosol fractions was independent of charcoal treatment. It was concluded that serum albumin with Zn2+ induces the activation of apoalkaline phosphatase due to Zn2+ binding.     

Putative role of cholesteryl ester transfer protein in removal of cholesteryl ester from vascular interstitium, studied in a model system in cell culture

A model system to study the putative role of cholesteryl ester transfer protein in the egress of interstitial cholesteryl ester is described. Confluent cultures of bovine aortic smooth muscle cells were labeled for 24 h with [3H]cholesteryl linoleyl ether and [14C]cholesteryl linoleate by incubation with bovine milk lipoprotein lipase. This method of labeling results in the transfer of cholesteryl linoleyl ether and cholesteryl ester to three compartments: a trypsin-releasable, trypsin-resistant and catabolic compartment (Stein, O., Halperin, G., Leitersdorf, E., Olivecrona, T. and Stein, Y. (1984) Biochim. Biophys. Acta 795, 47-59). The efflux of labeled cholesteryl linoleyl ether and cholesteryl ester from the extracellular and cell-surface related compartments into a serum-free culture medium containing 1% bovine serum albumin was studied during 24 h of postincubation. The efflux was expressed as a percentage of pulse value, i.e., radioactivity retained by the cell culture at the end of the labeling period. The efflux of [3H]cholesteryl linoleyl ether, [14C]cholesteryl ester and 14C-labeled free cholesterol (formed by cellular hydrolysis of cholesterol ester) into the culture medium with 1% bovine serum albumin was about 5% of the pulse value. Addition of human lipoprotein-deficient serum resulted in a 3-10-fold increase in the efflux of [3H]cholesteryl linoleyl ether and [14C]cholesteryl ester, but did not change markedly the efflux of 14C-labeled free cholesterol. Rat lipoprotein-deficient serum which does not contain cholesteryl ester transfer protein did not increase the efflux of [3H]cholesteryl linoleyl ether or [14C]cholesteryl ester. The rate of cholesteryl ester efflux in the presence of human lipoprotein-deficient serum was linear for about 6 h and increased further up to 24 h. Addition of Intralipid to medium containing human lipoprotein-deficient serum further enhanced the efflux of [3H]cholesteryl linoleyl ether and, to a lesser extent, that of cholesteryl ester. A similar effect was observed also by addition of rat VLDL to medium containing human lipoprotein-deficient serum. Inhibition of cholesteryl linoleyl ether and cholesteryl ester efflux and marked enhancement of free cholesterol efflux occurred when rat HDL was added to medium containing human lipoprotein-deficient serum, while human HDL was only slightly inhibitory. The results obtained with human lipoprotein-deficient serum were reproduced with partially purified cholesteryl ester transfer protein. Using the partially purified cholesteryl ester transfer protein, the efflux of cholesteryl linoleate was compared to that of cholesteryl oleate and was found to be the same.     

Mycoplasma growth factors in bovine serum fraction.

Mycoplasma growth factors in bovine serum fraction were separated by Sephadex G150 column chromatography and density ultracentrifugation. The major growth factor of bovine serum fraction eluted from the Sephadex column in the void volume. Its growth-supporting activity was greatly enhanced by the presence of bovine serum albumin in the mycoplasma culture media. Other investigators had previously identified the major growth factor in serum as an alpha-lipoprotein. Although density ultracentrifugation revealed the presence of traces of a high-density lipoprotein in bovine serum fraction, another, less dense component, isolated by ultracentrifugation (component 3) and containing cholesterol, cholesteryl esters, free fatty acids, triglycerides, and protein, but no lipoprotein, exhibited considerably more growth-supporting activity than did the high-density lipoprotein, thus indicating that at least two mycoplasma species do not require intact serum lipoprotein for growth. Both the high-density lipoprotein and component 3 exhibited maximum activity only in the presence of bovine serum albumin. A chloroform extract containing component 3 lipids combined with bovine serum albumin to form an effective, partially defined, less complex substitute for serum in mycoplasma culture media.     

Adhesion of enterotoxigenic Escherichia coli to immobolized intestinal mucosal preparations: a model for adhesion to mucosal surface components

The ability of enterotoxigenic strains of E. coli to adhere to immobilized mucosal components prepared from the large and small intestines of mice was examined in vitro. Various strains of E. coli were labeled with (³H)-acetate and incubated in tissue culture plates containing immobilized mucosal components or bovine serum albumin. E. coli strains which were positive for K88 or K99 antigen, and one E. coli strain isolated from a human urinary tract infection, were shown to adhere readily to large and small intestinal mucosal preparations, but not to bovine serum albumin. E. coli K-12 and a variety of enterotoxigenic strains isolated from humans, including a CFA/1 positive strain, demonstrated little or no ability to adhere to any of the preparations. E. coli adhesion to the mucosal preparations was shown to be mannose-resistant for all E. coli strains tested, but was inhibited by growth of the organisms at 18°C. Adhesion of each of the K88 or K99 positive strains was inhibited by homologous antiserum, but not by heterologous antiserum or normal rabbit serum. The data indicate that the mucosal preparations employed contain receptors for specific bacterial adhesins, and suggest that the use of such preparations may provide an alternative to the use of whole cells both as a source of receptor and as a means of investigating the adhesive properties of E. coli.     

Difference in growth factor requirements of rat 3Y1 cells among growth in mass culture, clonal growth in low density culture, and stimulation to enter S phase in resting culture

A semiserum-free medium was developed for monolayer culture of rat 3Y1 fibroblastic cells. The main components of the developed medium added to Dulbecco's modified Eagle's medium (DMEM) were insulin, transferrin, epidermal growth factor, poly-D-lysine, bovine albumin, oleic acid, and bovine alpha-globulin. In this medium, 3Y1 cells grew in mass culture at much the same rate as in DMEM supplemented with 10% fetal bovine serum (FBS), and colonies, albeit of smaller sizes, did form. Virally transformed derivatives of 3Y1 (simian virus 40-3Y1, polyoma virus-3Y1 and adenovirus type 12-3Y1) also formed colonies in the semiserum-free medium. When trypsinized 3Y1 cells were seeded with the medium lacking alpha-globulin, neither growth in the mass culture nor clonal growth in the low density culture (clonal growth) occurred. In this case, cell spreading was inhibited by albumin, and this inhibition was overcome by adding alpha-globulin or treating dishes with serum. When albumin was excluded from the semiserum-free medium, clonal growth did not occur, whereas growth in mass culture and stimulation of DNA synthesis in the resting mass culture (stimulation of DNA synthesis) were not so drastically affected. When oleic acid was removed, growth in mass culture was inhibited considerably, but no considerable effect was seen on clonal growth or on stimulation of DNA synthesis. In the absence of insulin, stimulation of DNA synthesis was inhibited more markedly than when other components were removed, but such was not the case with growth in mass culture and clonal growth.     

Ability of melanins to protect against the radiolysis of thymine and thymidine

Individuals with black skin rarely get skin cancer, and melanomas, tumors arising from pigmented cells, are generally resistant to radiation therapy. The role of melanin in these two phenomena has not been defined, but oxygen-radical species have been implicated in both effects. These studies were undertaken to determine the ability of various melanins to compete for ionizing radiation-produced radicals which destroy nucleic acid bases. The ability of Sigma eumelanin (S-eumelanin) to protect against the radiolysis of thymidine in buffered solutions was compared to the protective ability of seven amino acids, including melanin precursors; bovine serum albumin, as a model protein; ficoll, as a model polysaccharide; and DNA. Both proteins and polysaccharides are known to scavenge hydroxyl radicals in cells. The concentration of thymidine after exposure to gamma radiation was determined by High Performance Liquid Chromatography (HPLC) analysis after removal of insoluble melanin by acid precipitation. S-eumelanin was more effective at competing with thymidine for free radicals than bovine serum albumin, Ficoll, or DNA, but less effective than certain of the small molecules. Several of the above compounds were also examined for ability to protect against thymine radiolysis. In addition, melanins from other sources were compared to S-eumelanin. Of these, enzymatically synthesized phaeomelanin was the most effective. The results indicate that melanins can compete for base- and nucleoside-damaging free radicals more effectively than other cellular macromolecules. Of the small molecules, the phenolic compounds had the greatest scavenging ability. In vivo, melanins are found in melanosomes bound to protein. Therefore, the relevance of these findings to the photo- and radiobiology of melanins in vivo has yet to be determined.     

Increased phagocytosis of outer segments in the presence of serum by cultured normal, but not dystrophic, rat retinal pigment epithelium

Cultured normal rat retinal pigment epithelium (PE) ingested six times more rod outer segments in the presence of 20% fetal bovine serum than in serum-free medium. PE cultured from Royal College of Surgeons (RCS) rats with hereditary retinal dystrophy, known to have a defect in vivo in the phagocytosis of shed outer segment tips, ingested amounts of outer segments comparable to normal PE in serum-free medium but did not show an increase in the presence of serum. In both strains of rat PE phagocytosis of latex spheres was similar in the absence of serum and was six-fold higher in the presence of serum, showing that the RCS phagocytic deficiency for outer segments in vitro is not due to a general defect in the phagocytic capacity of the cell. Increased phagocytosis of outer segments by normal PE was observed in the presence of the high molecular weight fraction of ultrafiltered serum but was not seen with serum that was heated at 93 degrees C or precipitated with 5% trichloroacetic acid. Bovine serum albumin had no effect on phagocytosis. These results are consistent with the idea that the phagocytosis of outer segments by cultured normal rat PE, but not by cultured RCS rat PE, is increased in the presence of a specific protein or other macromolecular component of fetal bovine serum.     

Production of extracellular polysaccharides by Lactobacillus delbrueckii ssp. bulgaricus NCFB 2772 grown in a chemically defined medium

Lactobacillus delbrueckii ssp. bulgaricus NCFB 2772 produced an extracellular polysaccharide when grown in a chemically defined medium with glucose or lactose as the substrate carbohydrate. The isolated extracellular polysaccharide had a sugar composition of glucose, galactose and rhamnose in a ratio of 1:6.8:0.7. The production of extracellular polysaccharides increased at higher temperatures, but the bacterium rapidly lost its polysaccharide producing ability at 47°C. Production of polysaccharides was growth-related: no polysaccharide production was found after growth had ceased. An excess carbohydrate did not result in increased polysaccharide production.     

Continuous culture of rat C6 glioma in serum-free medium

In this communication we describe serum-free culture conditions for the serial propagation of the C6 glioma cell line. The growth rate, saturation density, and morphology of these cells are equivalent to those of their serum-grown counterparts when cultured in a 3:1 mixture of Dulbecco's modified Eagle's medium and Ham's medium F-12 supplemented with trace elements, insulin, transferrin, fibroblast growth factor, linoleic acid complexed to fatty acid-free bovine serum albumin, and a serum-spreading factor (SSF) partially purified from human plasma. The requirement for SSF in the medium can be satisfied by preincubating the tissue culture dishes with SSF. Tissue culture dishes sequentially pretreated with poly-D-lysine and purified cold insouluble globulin will also substitute for this requirement. The fatty acid-free bovine serum albumin/linoleic acid complex increases the growth rate of these cells but has no appreciable effect on their morphology, saturation density, or ability to grow with repeated subculture. The growth stimulation caused by this complex appears to be dependent on the fatty acid, as the fatty acid-free bovine serum albumin alone has no effect on the growth rate. Linoleic acid is cytotoxic in the absence of bovine serum albumin, and the fatty acid-free bovine serum albumin prevents this toxicity. Other fatty acids including oleic, arachidonic, and palmitic only partially substitute for the growth-promoting effect of linoleic acid.     

Cytotoxicity of thermally oxidized fats

Summary The effects of oxidized fat components (free fatty acids from the distillable nonurea adductable fraction) isolated from heated corn oil or heated olive oil on the morphology and growth of heart cells in primary culture were investigated. The free fatty acid fractions isolated from the fresh fats served as controls. Different concentrations of the fat fractions (20, 60, and 100 μg/ml) were added to the medium in the form of an emulsion with bovine serum albumin (Fraction V, poor in unesterified fatty acids). In the cell cultures treated with heated fats, intracellular lipid accumulation, increased cytoplasmic vacuolization, mitotic aberrations, pyknotic cells, and decreased mitosis were observed and were more pronounced in the case of the heated olive oil. These cytotoxic effects increased with higher concentrations of heated fats in the medium. The fresh fats also produced intracellular lipid accumulation, reductions in mitosis, and changes in the nucleus and cytoplasm, at the higher levels. These effects were greater in fresh olive oil-treated cultures. These observations indicate that oxidized fat components interfere physically or biochemically with normal cell functions resulting in pathological changes.